A Prenylation Motif Is Required for Plasma Membrane Localization ...

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Jul 29, 2016 - HA-Yck2p immunoreactivity associates with the particulate fraction. ..... in mammalian cells. Walworth for helpful advice, Mike Wigler and David G. Drubin for gifts .... Lerner, R. A,, and Wigler, M. (1988) Mol. Cell. Biol. 8,2159- ...
Vol. 269, No. 30, Issue of July 29, pp. 19271-19278, 1994

THE JOURNALOF nroLmlCAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biolou, Inc

Printed in U.S.A.

A Prenylation Motif Is Required for Plasma Membrane Localization and Biochemical Function of Casein Kinase I in Budding Yeast* (Received for publication, March 2, 1994, and in revised form, May 10, 1994)

Ales Vancura, Anna Sessler, Betty Leichus,and Jeff KuretS From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2220 tion alters the activityof each of these substrates and is necessary for full originunwinding activity of T-antigen. Although an important role for CK1 in cell regulation has long been suggested on the basis of these phosphorylations, definitive proof that CK1 acts on these proteins in vivo is lacking. or what itsrole Although it is not clear how CK1 is regulated is within thecell, several studies have identified where itfunctions. For example, mammalian CKIashows cell cycle-dependent localization to mitotic spindles (161, implicating theenzyme in mitosis. In addition, mammalianCK1 activity is stimulated by insulin, interleukin-1, and tumor necrosis factor in a dosedependent fashion (17-19), placingaCK1 isoform atthe plasma membrane, where it may interact withphosphatidylinositol 4,5-bisphosphate (20, 21). The contradictory distribution of CK1 activity stems from the existence of multiple isoforms. Currently, 4 closely related gene products are recognized in mammals and are termed CKIa, -& -7, and -6 (4, 7). Additional forms and splice variants may add to thecomplexity. We have turned tolower eukaryotes to facilitate the detailed study of individual CK1 isoforms (5, 22). In budding yeast, three forms of CK1 are currentlyrecognized. Two of these (enCasein kinase I (CK1)’ is a protein kinase common t o all coded by YCKl and YCK2) form an essential gene pair whose eukaryotic cells (1,2). Once considered a singleentity, it is now products are very similar in structure (5, 6) and contain a known t o consist of multiple isoforms that togethercomprise a consensus sequence for prenylation at their C termini (Glydistinct branch of the eukaryotic protein kinasefamily (3-8). Cys-Cys). Two phenotypes are associated with these kinases. Family membersidentified todate consist of a highlyconserved First, theiroverexpression results inhalotolerance, suggesting that CK1 may function in the osmolarity signaling pathwayof N-terminal catalytic domain joined to a variable C-terminal region that isnot conserved between familymembers and that budding yeast (23). Second, yck- cells show abnormalities in is variable in length. The catalyticdomain is characterizedby bud morphology and cytokinesis prior to growth arrest, sugits unique primary structure (5) and by an unusual substrate gesting a rolefor CK1 in thecontrol of cell growth polarity (24). Disruption of the third CK1 isoform (encoded by HRR25) reselectivity that is directed toward phosphate groups rather sults in x-ray and chemical mutagen sensitivity, consistent than unmodified amino acids (9, 10). Many proteins are in vitro substrates of CK1, including cy- with a role in the recombinational (or double-strand break) toskeletal proteins (myosin, ankyrin, troponin, spectrin, pro- DNA repair pathway (3). In addition, hrr25 loss-of-function tein 4.1, and neural filament proteins), proteins involved in mutants are sensitiveto benomyl, a microtubule depolymerizmRNA translation (tRNA synthetases and initiation factors 4B, ing agent. Previously, we showed that Yck2p is a particulate enzyme (5) 4E, and51, signaling molecules (insulin receptor, the regulatory and proposed that its C-terminal prenylation motif anchors it subunit of proteinphosphatase-1, erythrocyte aniontransporter), enzymes involved in nucleic acid metabolism (SV40 to intracellular membranes. Here we identify the subcellular T-antigen, RNA polymerase I and 11, and p53), and metabolic compartments containing Ycklp,Yckap, and Hrr25p, test our enzymes such as glycogen synthase andacetyl-CoAcarboxylase model of CKUmembrane association, and gauge the impor(1). However, correlation of CK1-specific phosphorylation sites tance of that association for in vivo function. with sites phosphorylated in vivo is available only for SV40 MATERIALS AND METHODS T-antigen ( l l ) , glycogen synthase (12, 13), and the transcripStrains and Media-Yeast strains used in this study (listed in Table tional regulators CREM (14) and p53 (15). CK1 phosphorylaI) were grown at 30 “C in rich medium (YF’D; 1%yeast extract, 2% 2% glucose) or underselection in synthetic minimal * This work was supported by Grant GM 42816 from the National Bacto-peptone, Institutes of Health. The costs of publication of this article were de- medium (SD) supplementedwithappropriate nutrients. Yeastwere frayed in part by the payment of page charges. This article must there- manipulated as described previously (5). Expression PZasmids-pJK435-YCK2 was constructed from pRS415 fore be hereby marked “aduertisement”in accordance with 18 U.S.C. (the pBluescript I1 version of pRS315; Ref. 25) through insertion of a Section 1734 solely to indicate this fact. ADHZ promoter, hemag.$ To whom correspondence and reprint requests should be addressed. 3.0-kilobase BamHI fragment containing the glutinin-epitope coding sequences (261, YCK2, and the ADHI terminator E-mail: [email protected]. (obtained from pAD5-CHI; Ref. 5). The final construct, which drives The abbreviations used are: CK1, casein kinase-1; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline;BSA, bovine the constitutive expression of Yck2p tagged at its N terminus with a serum albumin. 19-residuepeptide derived from the hemagglutinin antigen of influenza

The subcellular distribution of three casein kinase I (CK1)homologs, encoded by the YCKl, YCK2, and HRR25 genes, has been determined in budding yeast through a combination of subcellular fractionation and immunofluorescence methods. Both Yck proteins are tightly associated with the plasma membrane or underlying cytoskeleton and require both high-salt and nonionic detergent for extraction.Associationis mediated primarily by the prenylation motif foundat the C terminus of both Yck proteins. In contrast, the third CK1 homolog, Hrr25p, is found predominantlyin the nucleus and only partially in the plasma membrane. Despite partial colocalization with the Yck proteins, Hrr25p is unable to rescue the ycklAyck2A phenotype. However, achimeric kinase containing the N-terminal kinase domainof Hrr25p and the C-terminalregion of Yck2p contains full Yck activity in vivo. These datasuggest that members of the casein kinase I family have distinct but partially overlapping distributions in the cell that are mediated by their unique C-terminal regions.

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virus (26), was transformed intoW303 diploid or W303-la haploid cells to createstrains JK6and JK6-la (Table I). Expression plasmid pJK435HRR25 was derived frompJK435-YCK2 by replacing YCK2 with HRR25. HRR25 was prepared from genomic DNA through the polymerase chain reaction and modified to contain an NdeI site at the5'-end and anXhoI site at the 3'-end (5). Transformation of this plasmid into W303-la cells created strain JK8-la. To generate the yck2ACC allele, pJK435-YCK2 was mutagenized by the method of Kunkel et al. (27) using the oligonucleotide: 5"CGTTTTCTATTCTATCCTAGCTTACTG. The final construct, pJK435yckZACC, encodes epitope-tagged YckapACC, which lacks the C-terminal CC-motif of its full-length parent. Transformation of this plasmid into W303-lacells created strain JK10-la.The yck2AN allele was generated from pJK435-YCK2 with the mutagenic oligonucleotide: 5'-

(450 x g x 5 rnin), washed four times in ice-cold spheroplast buffer (2-mercaptoethanol was omitted), resuspended (to A,,, -= 50) in lysis buffer (20 mM triethanolamine acetic acid, pH 7.2, 0.8 M sorbitol, 1 mM EDTA, 1 m~ PMSF, and 20 mg/ml each of leupeptin, pepstatin, and aprotinin), homogenized with 20 strokes in glass-teflon homogenizer (Wheaton Scientific), and centrifuged (450 x g x 3 min). After the resultant pellets were re-extracted and centrifuged as above, the supernatants were pooled and taken as the total cell lysate. This was centrifuged sequentially at 5,000 x g x 10 min, 10,000 x g x 10 min, 25,000 x g x 10 min,and 170,000 x g x 1h to produce pellet fractions P1, P2, P3, and P4, respectively. Each pellet fraction was resuspended in 1 ml of sucrose solution (55% sucrose, 20 mM Tris-HC1, pH 7.5, 1mM EGTA, 1 mM PMSF, and 20 mgimleach of pepstatin, aprotinin, and leupeptin) for subsequent analysisor sucrose gradient fractionation. The supernatant GTCGCGTGAWGAGACATTT"GGkL4ACTA""l"I'C. from the 170,000 x g spin was taken as the soluble (SIfraction. The yck2-2" allele (24) also was created from pJK435-YCK2 by oliPellet fractions P 1 + P2 or P3 + P4 were combined, homogenized,and gonucleotide-directed mutagenesis. The resultant expression cassette subjected to sucrose gradient fractionation as described previously (34). (on a 3-kilobase BamHI fragment) was inserted into pRS414 (25). The Briefly, combinedpellet fractions were placed at thebottom of a 1.4 x 8.9 final construct, pJK434-yck2-2", encodes epitope-tagged Y~k2p-2~' and cm centrifuge tube and overlaid with the following volumes and conwas transformed into diploid JKlOl cells (5) to generate strain JK102. centrations of sucrose (containing 20 mM Tris-HCI, pH 7.5, 1mM EGTA, Following sporulation of JK102 cells and tetrad dissection, haploid seg- 1nm PMSF, and 20 mgiml each of pepstatin, aprotinin, and leupeptin) regants were selected that were HIS', URA', and TRP. One such strain at 0°C: 1ml x 50%, 1ml x 47.5%, 1.5 ml x 45%, 1.5 ml x 42%, 1.5 ml (JKlll), isycklAyck2A and contains pJK434-y~k2-2~ as only the copy of x 40%, 1ml x 37.5%, 1 ml x 35%, and 1ml x 30%. After centrifugation a YCK gene. J K l l l cells grow normally at 22-30 "C (permissive tem- (16 h x 170,000 x g; SW41 rotor), the gradients were fractionated from perature), but do not grow at 37 "C (nonpermissive temperature). the top (600-ml fractions) and labeled sequentially as fractions 1-19. To generate an Hrr25plYck2p chimera, YCK2 (blunt NdeIIApaI frag- The pellet was resuspended in 600 ml of sucrose solution and taken as ment) was inserted into the XhoI site of pJK435-HRR25, creating tan- fraction 20. Fractions were assayed in duplicate for protein (351, for dem HRR25 and YCK2 sequences. Nucleotide codons lying between standard marker enzymes, including vanadate-sensitive plasma memLeuzg3of Hrr25p and of Yck2p were then removed by deletion brane ATPase (36), cytochrome c reductase (37), cytochrome c oxidase mutagenesis (28) using the oligonucleotide: 5'-CCAACCACGGCCAC- (38), a-n-mannosidase (39), and GDPase (401, and for Yckap, Rasap, CAWCAACATTGTCCAATCGAACAAG. The resultant plasmid, actin, and cofilin immunoreactivity as described below. pJK435-HRR25/YCKZ,drives the expression of a chimeric kinase conSolubilization of YckZp-Gradient fractions containing epitopesisting of the catalytic domain of Hrr25p (residuesMet1-Leuzg3) fused to tagged Yck2p were pooled, diluted 3-fold (in 20 mM Tris-HC1, pH 7.5, 1 the C-terminal region of Yck2p (residues A ~ n ~ ~ - C y s ' ~ ~ ) . mM PMSF, and 20 mgiml each of leupeptin, aprotinin, and pepstatin), and divided into 8 aliquots. Individual aliquots were incubated (15 min Antibodies-Polyclonal antibodies were raised in mice against purified, recombinant Cki1"298 from fission yeast (29). The IgG fraction was on ice) with the following reagents: 1% Triton X-100, 1 M NaC1, 1% purified on a column of Protein A-Sepharose CL-4B (Pharmacia Bio- Triton X-100+ 1 M NaC1, 6 M urea, 1% SDS, 0.2 M Na,CO,, pH 11, and tech), and anti-Ckil antibodies were affinity-purified on nitrocellulose 1 M NH,OH (in this case the incubation was 30 min at 37 "C). After filters preadsorbed with Ckil as described previously (30). Antibodies centrifugation (2 h x 170,000 x g), the distribution of Yck2p in the against Ras2p (31), actin, and cofilin (32) were the gifts of the M. Wigler resultant particulate and soluble fractions was assayed by Western analysis. and D. G. Drubin laboratories, respectively. Density-shift Purification of Plasma Membrane Vesicles-Plasma Immunofluorescence-Yeast cells were grown to early log phase in membranes were separated from intracellular membranes throughlecSD medium and fixed in situ by direct addition of paraformaldehyde solution (16% paraformaldehyde, 10 m~ potassium phosphate, pH 5.6) tin-induced modification of their density (41, 42). First, JK6-la cells to the medium (4% formaldehyde, final concentration). After shaking were grown and spheroplasted as described above.Second,exposed the mixture (2 h at room temperature), the cells were washed twice in glycoproteins on the surface of the intact spheroplasts were selectively phosphate buffer (10 m~ potassium phosphate pH 5.6) and once with coated with concanavalin A. Specifically, spheroplasts (680 A,,, units; spheroplast buffer (SB; 20 mM potassium phosphate, pH7.5, 1.4 M 1.38 g wet weight) were resuspended in 10 ml of BufferA (1.4 M sorbitol, 40 mM potassium phosphate, pH 7.5,5 mM MgSO,) and divided into two sorbitol, 10 mM NaN,, 0.3% 2-mercaptoethanol). To prepare spheroplasts, fxed cells were resuspended in SB (A,, = equal aliquots. One aliquot was incubated with concanavalin A (1 mgi 100) and digested with Zymolyase 100-T (0.5 mglml; ICN Immunobio- ml) for 15 min a t room temperature, the other was untreated. To remove logicals) a t room temperature until at least 75%of the cells were con- excess lectin, spheroplasts were washed three times with cold Buffer A. verted to spheroplasts (33). The resultant spheroplasts were washed Next, lectin-bound spheroplasts were lysed in 5 ml of homogenization three times in ice-cold SB andresuspended in SB without 2-mercapto- buffer (20 m~ Tris-HC1, pH 7.5, 5 mM MgSO,, 1 mM PMSF, 1 mgiml ethanol. The addition of protease inhibitors broughtthe suspension to 1 DNase, and 20mgiml each of pepstatin, aprotinin, and leupeptin), mM PMSF and 20 mg/ml each of leupeptin, pepstatin, and aprotinin. homogenized with 20 strokes in a 10-ml glass-teflon homogenizer, and Spheroplasts were immobilized on poly-L-lysine-coated coverslips, centrifuged (450 x g x 3 min) to remove unbroken cells. After the resultant pellets were re-extracted and centrifuged as above, the superwashed twice in SB-containing protease inhibitors, fixed in cold methanatants were pooled and taken as the total cell lysate. Finally, cell nol (5 min at -20 "C), and blocked (4 x 10 min) in PBS supplemented lysates were fractionated on the basis of density by centrifugation (30 with protease inhibitors, 1% BSA, and 1% normal goat serum. To label Yck proteins, coverslips were incubated with primary anti- min at 250 x g) through 10-ml cushions of 1.4 M sorbitol. The material remaining atop the sorbitol cushion was harvested by body (affinity-purified mouse anti-Ckil antibodies or mousecontrol IgG diluted in PBS supplemented with protease inhibitor, 1%BSA, 1% ultracentrifugation (1 h x 170,000 x g) and taken as the crude memnormal goat serum) for 1h. Unbound antibodies were removed by 8 x 10 brane fraction. When prepared from untreated yeast spheroplasts, this min washes with PBS (containing protease inhibitors, 1% BSA, 1% fraction contains the plasma membrane fraction and other membranebound organelles. However, coating the surface of yeast spheroplasts normal goat serum). To detect labeled Yck proteins, coverslips were incubated (1h at room with dense lectin molecules results in anincrease in the density of the temperature) with fluorescein-tagged goat anti-mouse IgG (CappeV plasma membrane, so that it no longer floats on the sorbitol cushion, Organon Teknika) at 60-fold dilution in PBS containing protease inhibi- but pellets along with high density material. This material was selectors and 1% BSA. Excess antibody was removed by washes (6 x 10 min) tively enriched by resuspending the pellet fraction in 10 ml of Buffer B in PBS containing protease inhibitors. Coverslips were drained and (in 20 mM Tris-HC1, pH7.5,l mM EDTA, 1nm PMSF, and 20 mgiml each prepared for microscopy in mounting medium (p-phenylenediamine, 1 of leupeptin, pepstatin, aprotinin) containing 1 M a-methylmannoside, homogenizing in a glass-teflon homogenizer to break up aggregates and mgiml in 90% glycerol, pH 8.0). Cells were viewed and photographed through a lOOx objective mounted on a Nikon Microphot FXA micro- promote the formation of vesicles, and incubating for 30 min at room temperature. Under these conditions, the cy-methylmannosidebinds the scope. lectin, releasingthe plasma membrane vesicles. After centrifugation at Subcellular Fractionation-Cells were grown under selection to A,, 1.5, harvested by centrifugation (2000 x g x 5 rnin), and sphero- 450 x g for 3 min to remove aggregates, the resultant pellets were re-extracted and centrifuged twice more, and the supernatants were plasted as described above. The resultant spheroplasts were centrifuged

Budding Yeast Casein Kinase I M

I

2

3

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TABLEI Yeast strains used in this study

4

130 -

Strain

-

W303

50W303-la

39 -

JK5

27 -

FIG.1.Immunodetection ofYcklp and Yck2p. Whole-cell lysates were prepared and subjectedto Western analysis withpolyclonal antiCkil antibody as described under “Materials and Methods.” Lane M, prestained markers phosphorylase b (130 kDa),bovine serum albumin (80 kDa), ovalbumin (50 kDa), carbonic anhydrase (39 kDa), andsoybean trypsin inhibitor (27 m a ) ; lanes 1 and 3, wild-type (W303-la) cells; lane 2, ycklAcells (JK21-la); lane4, yck2A cells (JK11-la).

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JK6 JK6-la JK8-la JK10-la JK11-la JK21-la JKlOl JK102 JKlll

Genotype

Source”

MATa his3-11,15 ade2-1 leu2-3, 112 t r p l - 1 u r d - 1ssdl-d2 canl-100 MATa ade2-1 his3-11,15leu2-3, 112 trpl-1 ura3-1 ssdl-d2canl-100 MATa ade2-1 his3-11,15 leu2-3, 112 trpl-1 ura3-1 ssdl-d2 canl-100 W303 [pAD5-YCK21 W303 [piK435-YCK2] W303-la [pJK435-YCIL21 W303-la tpJK435-HRR251 W303-la [pJK435-yck2ACCl W303-la yck2::URAJ W303-la yckl::HIS3 W303 yck2::URA3lyck2::URA3 yckl::HISSl+ W303 yck2::URA3/yck2::URA3 ychl::HIS31+ [pJK434-yck2-2-1 W303-la yck2::URAS yckl::HIS3 [pJK434-y~k2-2”]

1

2

2 3 3 3

3 2 2 2

3

3

pooled, concentrated by centrifugation (1h x 170,000 x g ) , resuspended Sources: 1, Rothstein laboratory (Columbia University,New York, in Buffer B containing 0.8 M sorbitol, and taken as the plasmamem- N Y ) ; 2, Ref. 5; 3, this study. brane vesicle fraction. Western Analysis-Aliquots of individual fractions were electrophoreJK6-la cells were fractionatedby differential gradient centrifused through polyacrylamide gels (9% for analysis of Yck proteins; 14% for analysis of cofilin) and transferred to nitrocellulose membranes as gation and subjected to Western analysis as described under “Materialsand Methods.” Although monoclonal antibody describedpreviously(5).Epitope-taggedproteinswerelabeledwith monoclonal antibody 12CA5 (26), whereas the Yck proteins, Rasap, 12CA5 was employed for clear quantitationofYck2p levels (51, cofilin, and actin were labeled by the antibodies described above. Im- qualitatively similar resultswere obtained with thepolyclonal munoreactivity was detected either by colorimetric assay using antianti-Ckil antiserum (data not shown). Results are shown in mouse IgG conjugated to alkalinephosphatase (5) or by enhanced Fig. 2. Consistent with previous experiments (51,nearly all chemiluminescence using sheep anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase (Amersham). Images were collected on HA-Yck2p immunoreactivity associates with the particulate x-ray film and quantifiedby laser densitometry (Molecular Dynamics). fraction. Approximately 85% of all HA-Yck2p sediments with

the P3 and P4 fractions, with themajority (71%) appearing in the P3 fraction. Again, qualitatively similar results were obDetection andQuantitation of Yck Proteins in Budding tained with the polyclonal anti-Ckil antiserum, demonstrating that this distribution is shared by both Ycklp andYck2p (data Yeast-Previously, in an effort to characterize the four CK1 isoforms of fission yeast, polyclonal antibodies were prepared not shown). We conclude, therefore, that Ycklp andYck2p coagainst the catalytic domain of Ckil (Ckil””*), the fission localize to a discreet, insoluble fraction of the cell that sediyeast homolog of the Yck proteins.’ In addition to cross-reacting ments with the P3 fraction. Clearly, neither Yck protein is with Cki2, these antibodies recognized a closely spaced 62-kDa nuclear. To increase the resolution of the analysis, combined P3 and doublet after Western analysis of budding yeast cell lysates (Fig. 1). Because Ycklp and Yck2p share -68% identity with P4 fractions werefurther purified on sucrose densitygradients Ckil through the catalytic domain and havemolecular masses and assayed for Ycklp, Yck2p, and HA-Yck2p. Under these of 62 kDa, i t seemed likely they comprised the protein doublet conditions, Yck immunoreactivity fractionates asa sharp peak in themiddle of the gradient, with the familiar 62-kDa protein recognized by theantiserum. To confirm thisassignment, strains containing null alleles of yckl (JK21-la) and yck2 doublet that corresponds to endogenous Ycklp and Yck2p co(JK11-la) were grown and subjected to Western analysis as migrating with the64-kDa HA-Yck2p (Fig. 3). From these reYck2p co-localize to the same described under “Materials and Methods.” The results, which sults we conclude that Ycklp and are illustrated in Fig. 1, prove that the upper, more intense subcellular fraction, and that, on the basis of their buoyant band of the doublet corresponds to Yck2p, whereas the lower density, the fraction is membranous (33).Although HA-Yck2p band corresponds to Ycklp.We conclude that both YCK genes is expressed from a heterologous promoter, it nonetheless loare expressed in budding yeast, and that their gene products calizes identically with authentic Yck2p and, therefore, is useare recognized selectively by our polyclonal antiserum. Assum- ful for analyzing Yck2p subcellular distribution and biological ing it reacts equally well with each of the Yck isoforms (which function. Yck2p Associates Exclusively with the Plasma Memis likely becausethe Yck proteins share>94% identity through the catalytic domain; Ref. 5),Yck2p is -6-fold more abundant brane-To characterize the membrane fraction harboring the Yck proteins, density gradient fractions were assayed for orthan Ycklp. To follow Yck2p with greater selectivity and sensitivity, the ganelle-specific enzyme markers as described under ”Materials enzyme was tagged with the influenza hemagglutinin epitope and Methods.” The results reveal that the peak of Yck immunoreactivity (illustrated in Fig. 3) co-migrates with the vana( H A ) and expressed from the ADHI promoter on a low copy plasmid as described under “Materials and Methods.” The re- date-sensitive ATPase, a classic marker for the plasma memsultant strain, JK6-la (Table I), allows specific detection of brane (data notshown). In fact, fractions 10-12, which contain epitope-tagged Yck2p (HA-Yck2p) with monoclonal antibody the peak of Yckp immunoreactivity, have a 23-fold higher specific activity for the ATPase than the crude lysate and a 14-fold 12CA5 (5,26). Ycklp and Yck2p Are CytoplasmicEnzymes-To determine higher specific activity than thesample loaded on the gradient fractions). Yckp immunoreactivity the subcellular distribution of the Yck proteins, lysates of (i.e. the combined P3 and P4 also co-migrated with a second plasma membrane-associated protein, Ras2p (Fig. 4). In contrast, the Yck proteins do not P.C. Wang and J. Kuret, unpublished data. RESULTS

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vesicles and furtherpurified on the basisof density. The plasma membrane vesicle fraction was prepared from JK6-la cells in thepresence and absence of concanavalin A and assayed for HA-Yck2p and Ras2p immunoreactivity and for marker enzyme activity as described above. The results,which 30are summarized inTable 11, show that Ras2p, a plasma membrane marker, is enriched 30-fold when plasma membrane vesicles are prepared in thepresence of lectin. Similarly, HAYck2p is enriched 24-fold under these conditions. In contrast, lectin treatment does not enrich thevacuolar (a-mannosidase), mitochondrial (cytochrome c oxidase),or Golgi (GDPase) markP1 P2 P3 P4 s Fraction ers. Themodest (1.6-fold) enrichment of endoplasmic reticulum FIG.2. Differential centrifugation analysis of Yck2p. JK6-la (NADPH-cytochrome c reductase) may result from the physical cells were fractionatedby differential centrifugationas described under association of a portion of this membrane system with the “Materials and Methods.” The amounts of epitope-tagged Yck2p in the resultant Pl(5,OOO x g),P2 (10,000 x g),P3 (25,000 x g),P4 (170,000 x plasma membrane (43). We conclude that theYck proteins asmembrane g ) , and S (soluble) fractions were determined by quantitative Western sociate primarily, if not exclusively, with the plasma blot analysis using the12CA5 antibody. Recovery refers to the percent- of yeast cells. age of total lysate HA-Yck2p immunoreactivity found in each fraction. Yck2p Is Tightly Associated with the Plasma Membrane-To further investigate the interactionbetween HA-Yck2p and the M 1 2 3 4 5 plasma membrane,conditions necessary for its extraction from gradient-purified membranes (i.e. fractions 10-13, Fig. 3) were established. Membranes were extracted with various agents, centrifuged at high speed,and subjected to Western analysis to 130 determine the amountof HA-Yck2p in the resultant supernatant andpellet fractions. Results are illustrated in Fig. 6. HA75 Yck2p was not solubilized after treatment with high salt (1 M NaCl), chaotropic denaturant(6 M urea),high pH (0.2 M Na,C03, pH ll.O), or nucleases (DNase or RNase), and thus 50 does not fit the profile of a peripheral or extrinsic membrane protein (44). Furthermore, the enzyme was not solubilized by nonionic detergent (1% TritonX-100) or by hydroxylamine, an agent that cleaves thioester bonds (45). Thus, HA-Yck2p does 39 not behave like an integral membraneprotein or like a protein FIG.3. YcklpandYck2p co-localize to intracellular m e m - associated with membranes through fatty acylation, such as branes. The P3 and P4 fractions prepared from JK6-la cells were palmitoyl groups. In contrast, a combination of salt and noncombined and subjected to density gradient centrifugation as described ionic detergent or the use of 1% SDS resulted in complete under “Materials and Methods.” After centrifugation to equilibrium, the gradients were fractionated from the top (fraction 1) to the bottom solubilization of HA-Yck2p. Weconclude that both hydrophobic (fraction 20) and subjected to Western analysis. The polyclonal anti- and polar interactions mediate the association ofHA-Yck2p Ckil antibody was used to detect endogenous Yck proteins (lowerdou- and the plasma membrane. The extraction profile described blet) and epitope-tagged Yck2p (upper singlet band) in each fraction. above suggests thatYck proteins may bind the plasma memLane M, prestained markers as in Fig. 1; lane 1, fraction 9;lane 2, fraction 11;lane 3, fraction 12; lane 4, fraction 13; and lane 5,fraction brane indirectly or in tandem withaccessory proteins. Localization ofYckby Immunofluorescence-In an effort to 15. HA-Yck2p co-migrates through the gradient with endogenous Yck protein and so appears to localize properly. characterize subcellular distribution at higher resolution, indirect immunofluorescence of the Yck proteins was analyzed in co-migrate with markerenzymes for mitochondria (cytochrome diploid strains W303 and JK6 as described under “Materials and Methods.” Because treatment of yeast cells with monoc oxidase), endoplasmic reticulum (NADPH-cytochrome c reductase), Golgi apparatus (GDPase), or vacuole (a-mannosi- clonal antibody 12CA5 consistently produced an unacceptable dase; Fig. 4). Density gradient fractionation of combined P1 background of fluorescence, the anti-Ckilpolyclonal antiserum and P2 pellets yielded qualitatively identical results: Yck im- was used to visualize both Yck proteins, with purified preimmunoreactivity co-fractionated withthevanadate-sensitive mune IgG serving as control. When W303 cells were treated with anti-Ckil antibodies, a ATPase (data not shown). Therefore,we conclude that theYck proteins associate with either the plasma membrane or with a weak punctate staining pattern wasobserved, with stain conmembrane fraction of similar buoyant density. centrated at the periphery (data not shown). The overexpresTo distinguish between these possibilities, plasma mem- sion strain JK6 yielded a similar pattern of immunofluoresintensity. In budding branes were purified by a n independent method and assayed cence (Fig. 7a),but with greater staining for the presence of Yck immunoreactivity. The strategy is de- JK6 cells, staining clustered around the neck region. In conscribed under “Materials andMethods” and illustrated inFig. trast, futed JK6 cells treated with the same concentration of 5. First, cell surface glycoproteins are exposed by digestion of control preimmune IgG showed only weak background staining the cell wall and then coated with the lectin concanavalin A. (Fig. 7b). We conclude that Yck proteins are not distributed The plasma membrane is selectively labeled under these con- uniformly throughout the plasmamembrane, but arefound in discreet locations, presumably along its cytoplasmic face. The ditions, because the cells are intact, and the lectin cannot gain access to the cell interior. Once the spheroplasts are lysed, punctate staining patternresembles that observed for compolectin-bound material is separated from other cellular mem- nents of the cortical cytoskeleton (e.g. Ref. 32). branes (total cell membranes) on the basis of its increased The CC-motifIs Required for Proper Localization of Yck2pdensity. After elution of the lectin with a competitive ligand BothYck proteins containa consensus sequence for prenylation (a-methylmannoside), the plasma membrane is sheared into at their C termini (theCC-motif). In otherCC-motif-containing

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Mannosidase GDPase

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8

p! 20

FIG.4. The Yck proteins co-migrate with the plasma membrane fraction. The P3 and P4 fractions from JK6-la cells were prepared, combined, and subjected to density gradient centrifugation as described under “Materials and Methods.” The resultant gradients were fractionated from top (fraction 1) to bottom (fraction 20) and assayed for HA-Yck2p and selected marker enzymes. Therecoveries of HA-Yck2p and Ras2p in each fraction are shown as percentage a of the total amounts loaded onto the gradient. The activities of cytochrome c oxidase and GDPasearepresented as totalactivity per fractionin unitsof nanomoles of product produced per min, whereas the activities of a-mannosidase and NADPH-cytochrome c reductase are presented units in of millimoles of product produced per min. Protein content and refractive index also are shown.

-

2

a9

$

n IO

10

cy

Y

f =

2

-4

a

2

300

3 , fn

200

2

m

0

E 1 5

s

B

o

0 0

10

0

0

20

Fraction

-

10

10

20

Fraction

NADPHCytcreductase

0

‘0 -5

17

20

I

-C

0

I

Refraction Index

10

. ”.

20

Fraction

Cell wall Organelles

Fracti TABLEI1 Density-shift purificationof plasma membrane vesicles Marker

I

Spheroplast I

Yck2p Ras2p a-Mannosidase GDPase Cytochrome c oxidase NADPH cytochrome c reductase

Plasma membrane vesicles Purification“

Recoveryb

-fold

%

24.0 30.0 1.0 1.0 1.2 1.6

56 63 7 5 8 14

a Purification is the ratio of total activity (or immunoreactivity) recovered in the plasma membrane vesicle fraction prepared with concanavalin A to activity recovered without it. * Recovery is the amountof each marker found in the purified vesicle fraction relative to the amount of each marker in totalcell lysates.

0 0 0 00

Concanavalin A Plasma membrane ghosts Elution of concanavalin A with o( - methylmannosids homogenization, differential centrifugation

d?;

- Plasmamembranevesicles

FIG.5. Isolation of plasma membrane vesicles from budding yeast.

proteins, such as the small GTP-binding proteins Yptlp and Sec4p, this segment mediates membrane association (46-48). To determine whetherthis is true for the Yck proteins aswell, a C-terminal deletion mutant CYck2pACC) was prepared as described under“Materialsand Methods,” expressed in JK10-la cells, and localized by subcellular fractionation. Upon differential centrifugation, the majorityof Yck2pACC pelleted withP4fraction (73%), whereastheremainderappeared were subsoluble. When combined pellets (Pl, P2, P3, and P4) 95%of jected tosucrose gradient centrifugation, approximately

Yck2pACC was found near thebottom of the gradient (fractions 16-19), with the remnant migrating at the same position as full-length HA-Yck2p (Fig. 8).We conclude that theCC-motif is the principal feature responsible for targeting Yck proteins to the plasma membrane, and that, in itsabsence, Yck proteins bind inappropriately to some other particulate fraction. To characterize the interaction between HA-Yck2pACC and this particulate material, gradient fractions 16-20 were combined and subjected to solubilization trials as described above for HA-Yck2p. Unlike HA-YcMp, however, nearly 80% of HAYck2pACC was solubilized with 0.3 M NaCl, whereas only 45% was extracted withTriton X-100 (data not shown). Becausethe Yck proteins are highly charged molecules, we suspect that under conditions of low ionic strength, such as those used for cell lysis and density gradient centrifugation, HA-Yck2pACC associateswith a particulatefractionunrelatedtoplasma membranes by nonspecific ionic interactions. Indeed, when JK10-la cells are lysed in thepresence of buffer containing 0.3 M NaCl, most HA-Yck2ACC is recovered in the supernatant. Under identical conditions, wild-type Yck2p is completely particulate. To determine whether plasma membrane association was required for in vivo function, the ability ofyck2ACC to comple-

Budding Yeast Kinase Casein

19276

I

ment the lethalityof ycklAyck2A double mutant strain JKlll gradients and assayed for HA-Hrr25p and organelle-specific (which harbors the temperature-sensitive ~ c k 2 - allele 2 ~ on a enzyme markers as described above. As shown in Fig. 11, low copy plasmid) was investigated.J K l l l transformants har- nearly all HA-Hrr25p in the P1P2fraction pellets at the botboring theplasmids pJK435-YCK2, pJK435-yck2ACC, or tom of gradient (fraction 20) along with genomic DNA (data not pJK435 vector alone were isolated at the permissive tempera- shown). Because it contains 53% of extracted HA-Hrr25p, but ture, replica-plated onto rich medium, and incubated a t t h e only 6% of total extractedprotein, this fraction does not consist nonpermissive temperature to test for complementation. Re- of unbroken cells. In contrast, when combined P 3 P 4 pellets sults are shown in Fig. 9. Whereas the vector alone was inac- werefractionated on sucrose gradients, HA-Hrr25p co-mitive, full-length YCK2 fully rescued the conditional lethality of grated with the plasma membrane marker, vanadate-sensitive strain JK111. Clearly, the Yck proteins perform an essential ATPase (Fig. 11).We conclude that Hrr25p and the Yck proidentical conditions, teins have distinct, yet partially function at the plasma membrane. Under overlapping,distributions. heterologous expression of yck2ACC also rescued the condi- Whereasthe Yck proteins associate exclusively with the tional lethal phenotype. We conclude that, although the CC- plasma membrane fraction, most Hrr25p co-migrates with inmotif is important for proper localization of YcMp, sufficient tact cell nuclei. its absence (-5% of kinase remainsat theplasma membrane in Because a portion of HA-Hrr25p co-localizes with Yck prothe total amount;Fig. 8) to rescue the ycklAyck2A phenotype. teins to the plasma membrane, we tested whether it could Localization of Hrr25p-Yeast cells contain a third CK1 complement the lethalityof ycklAyck2A double mutant strain homolog, termed Hrr25p, that regulates DNA repair (3). To JK111. As shown in Fig. 9, HRR25 was incapable of compledetermine if its intracellular distribution mirrored that of the mentation. The structural basis of this functional difference Yck proteins, cell fractionation experiments were performed as was examined through mutagenesis studies. First, we focussed described above. To facilitate detection, Hrr25p was tagged on the N termini of the Yck proteins, where a hydrophilic segwith the influenza hemagglutinin epitope and expressed from ment extends -70 amino acid residues from the catalytic dothe ADHZ promoter on low-copy plasmids as described under main. This featureis not found in Hrr25p or any other known “Materials andMethods.” The resultant strain, JK8-la(Table I), allows specific detection of epitope-tagged Hrr25p (HA- CK1 homolog (5). To examine its importance, an N-terminal Hrr25p) with monoclonal antibody 12CA5. To determine the deletion mutant of YcMp was prepared (pRS435-yck2AiV) and l for complementation. The resultantmusubcellular distributionof HA-Hrr25p, fractions P1, P2, P3, P4,assayed i n J K l lcells and S were prepared from JK8-la cells and subjected to West- tant, Yck2DNp, consists of the catalytic domain, the hydroern analysis asdescribed before. Results are shown in Fig. 10. philic tether region, and the CC-motif. As shown in Fig. 9, the As with the Yck proteins, essentially all HA-Hrr25p is associ- N-terminal region is not essentialfor Yck function, and, thereated with the particulate fraction. Unlike the Yck proteins, fore, its absence in Hrr25p is not the reason for its failure to however, most of it (-62%) sediments in the P1 and P2 frac- complement. Second, we examined the catalyticdomain of tions, with the remainderrecovered in P3 (24%) and P4(14%). Hrr25p, which shares -55% identity withYck2p. To determine To increase the resolution of the analysis, combined P 1 P 2 whether it retained the substrate specificity of Yck2p, a chiand P3P4 fractions were further purified on sucrose density meric kinasewascreated between the catalyticdomain of Hrr25p (residues 1-293) and the C-terminal domain of YcMp (residues 363-546) and assayed i n J K l l lcells for complemen100 L As shown in Fig. 9, thechimeric kinase wasactive in the tation. Q .P > assay. We conclude that the catalytic domains of Yck2p and E 80 5 s Hrr25pshare overlapping, although nonidentical, substrate E specificities, and that the unique, C-terminal regions of each 8 60 molecule control their access to substrates and regulators.

*

h

Y

40

DISCUSSION

Y 0

>; a

20

X

0

1

2

3

4

5

6

7

8

Extraction condition FIG.6. Yck2p is tightly associated with the plasma membrane. Sucrose gradient fractions containing HA-Yck2p were pooled (fractions 10-13; Fig. 31, divided into 8 aliquots, and incubated with the reagents listed below. The percentage of total HA-Yck2p immunoreactivity extracted from the pellet (P)into the soluble( S ) fraction was determined by quantitative Western analysis after high-speed centrifugation. Lune 1, control (no treatment);lune 2,6M urea; lane 3,0.2M Na,CO,, pH 11; lune 4 , l M NH,OH; lane 5,1% Triton X-100; lune 6 , l M NaC1; lune 7,1% Triton X-100 + 1M NaCl; lane 8, 1% SDS.

FIG.7. Intracellular localization of Yck proteins by indirect immunofluorescence. JK6 cells were processed for immunofluorescence a s described under “Materials and Methods” using affnity-purified anti-Ckil antibody ( a ) or a n equivalent concentrationof normal rabbit IgG ( b ) .

Sequestration of protein kinases to discreet cellular compartments can facilitateor restrict theiraccess to a limitedrange of substrate proteins.Proper spatial organization is especially important for CK1 homologs, which are invariably isolated as constitutively active phosphotransferases (1).Here we examined the subcellular distribution of two CK1 homologs from budding yeast (theYck protein kinases) in detail and compared i t to thatof a third homolog, Hrr25p. The resultsshow that the Yck2p is associated exclusively with plasma membrane or underlying cytoskeleton, and that theassociation is mediated by the prenylation motif Xaa-Cys-Cys. Because Ycklp co-localizes with Yck2p and functions similarly, we suspect that theproperties determined for Yck2p will apply to Ycklpas well.

Budding Yeast Casein Kinase I

19277

2 0

a I 0 O0

10

2o

r

P1

P2

P3

P4

s

Fraction Fraction FIG.8. Deletion of the CC-motif results in mislocalization of FIG.10. Differential centrifugation analysis of epitope-tagged Yck2p. JK6-la and JK10-la cells were grown, spheroplasted, lysed, Hrr25p. JK8-la cells were fractionatedby differential centrifugationas and fractionated into P1, P2, P3, and P4 fractions as described under describedunder"Materialsand Methods." Theamount of epitope"Materials and Methods." The resultant pellet fractions were pooled tagged Hrr25p in the resultant P1 (5,000 x g), P2 (10,000x g ) , P3 and subjected to density gradient centrifugation and quantitative West- (25,000 x g ) , P4 (170,000 xg), and S (soluble) fractions were determined ernanalysisusing 12CA5 monoclonal antibody. Recoveries of HA- by quantitative Western blot analysis using the 12CA5 antibody. ReYck2pACC and full-lengthHA-Yck2p are the percentageof total lysate covery of HA-Hrr25p is shown as the percentage of crude cell lysate immunoreactivity found in each fraction. immunoreactivity found in each fraction. A

B h

40

*e C

1 0

E

Y

2o

d)

In

m

n

2

FIG.9. S t r u c t u r a l f e a t u r e srequired f o r Yck function at 37 "C. 0 The ycklAyck2A double mutant strain J K l l l (which contains the temperature-sensitive y ~ h 2 - 2allele ~ on a lowcopy plasmid), harboring 0 10 20 pJK435 vector alone ( I ) , pJK435-YCK2 (2),pJK435-yck2ACC (3), Fraction pJK435-yck2W (41, pJK435-HRR25 ( 5 ) , and pJK435-HRR25/YCK2 FIG. 11. Hrr25p is primarily nuclear. Combined P l P 2 and P 3 P 4 chimera (6), was streaked onYPD plates and incubatedat thepermissive temperature (22"C) (A) or the nonpermissive temperature (37 "C) fractions from JK8-la cells were subjectedto density gradient centrifu( B ) .Only those constructs that complement theycklAyck2A defect grow gation a s described under "Materials and Methods." After centrifugation to equilibrium, gradients were fractionated from top (fraction 1) to at the nonpermissive temperature. bottom (fraction 20) and assayed for HA-Hrr25p and selected marker enzymes. Recovery of HA-Hrr25p is shown as the percentage of crude cell lysate immunoreactivity found in each fraction. The plasma memThe intracellular distributionof the Yck proteins was estab- branefractionismarked by vanadate-sensitive ATPase activity lished through three independentapproaches: density-equilib- (nanomoles/min).

rium purification of yeast organelles, purification of plasma membrane vesicles on the basis of their interaction with concanavalin A, and indirect immunofluorescence. Both cell subcellular fractionation approaches unambiguously place these proteins at the plasma membrane along with the vanadatesensitive ATPase and withRas2p. Consistent withour model of Yckp structure (5),membrane binding is mediated by a CCmotif found at the C terminus of each kinase. This motif is a substrate for BetSp, the catalytic subunit of the S. cerevisiae geranylgeranyl transferase I1 (48). Unlike other prenylated enzymes, however, the Yck proteins are notsolubilized by membrane-dispersing agents, suchas nonionic detergent. Clearly, a more complex interaction existsbetween Yckp and the plasma membrane fraction than simple insertion of an isoprenoid moiety into a lipid bilayer. In view of these results,it is possible that plasma membrane association is mediatedby multiple interactions. In the case of other prenylatedproteins, such as mammalianp21", a second signal in theform of a polybasic domain ( ~ 2 1 ~ -or ~ .palmitoyl*) ation site(p21H8"""and p21N.ms)is required for targeting to the plasma membrane (49, 50). However, these factors seem unlikely in the case of the Yck proteins, because palmitoylation sites or clustered basic residues are not found upstream of their CC-motifs (51,and because agents that cleave the thioester bonds found in fatty-acylated proteins do not releaseHA-Yck2p from the membrane.

A second possibility is that the putative prenyl groups of the Yck proteinsdirectly mediate protein-protein interactions rather than protein-membrane association. For example, farnesylated yeast Ras2p binds adenylate cyclase with 100-fold higher affinity than unmodified Ras2p (51). Although subsequent palmitoylation is required for efficient membrane localization, it has little effect on its interaction with adenylate cyclase. Similarly, deletion of the CC-motif from YcMp has no effect on protein kinase activity (as determined by assaying casein phosphorylation in immunoprecipitates; data not shown), but leads to mislocalization and partialloss of function in vivo. By analogy, prenylated Yck proteins may bind to their own plasma membrane-associated protein or receptor. Both the extraction and immunofluorescence data suggest that theYck proteins bind to an insoluble component of the plasma membrane fraction, rather thandiffusing randomly throughoutthe lipid bilayer. Indeed, the punctateimmunofluorescence pattern ofYck proteins resemblesthose of cortical actin andcofilin (32), components of the cytoskeleton that co-migrate with both Yck proteins through density gradients (datanot shown). Alternatively, the Yck proteins may associate with other membrane components. In the case of mammalian CK1, binding to the plasma membraneis mediated in partby glycophorin, an integral membrane protein (20). Yeast CK1 may interact with a homologous protein.

19278

Budding East Casein Kinase 1

What is the regulatory function of CKl? At this point we can only speculate, but in thecase of the Yck proteins, their intracellular location and associated phenotypes point toward a role in theregulation of the cytoplasmiccytoskeleton. In particular, the emerging relationship between budding yeast morphology, osmosensitivity, and its actin cytoskeleton suggests that Yck activity may affect the mobilization of the cortical cytoskeleton. First, overexpression of YCK2 results in halotolerance. Although this may arise through any of several different mechanisms (e.g. Ref. 23), an i n ~ ~ i possibility n g is that Yck2p facilitates the redistribution of actin filaments following osmotic challenge (52). Second, ~ckZAyckZAcells have multiple elongated buds, indicating a failure to switch to isotropic bud growth, and become multinucleate, indicating a failure in cytokinesis (24). Although these defects most closely resemble those observed after disruption of the @-subunitof a type 2A protein phosphatase (cdc55; Refs. 24 and 531, they also resemble many of the defects observed after failure to activate Clb/Cdc28 kinase in theG, phase of the cell cycle (54). Thus, the Yck kinases may function downstream of ClbICdc28 and participate in theswitch between apical and isotropic bud growth at thelevel of the cytoskeleton. In contrast, the predominantly nuclear localization of Hrr25p is consistent with its proposed role as a regulator of DNA metabolism (3).Unlike the Yck proteins, Hrr25p contains the sequence Thr-Lys-Lys-Gln-Lys-Tyr within its catalytic domain, which has been suggested to comprise a nuclear localization signal (3). Nonetheless, a significant portion (-35%) of Hrr25p cofractionates with the plasma membrane fraction in sucrose density gradients. It is not clear whether this represents a stable bimodal distribution or the reversible translocation of the enzyme among cellular compartments. Despite its association with the plasma membrane, Hrr25p is incapable of supplying the essential function of the Yck proteins. The failure is unrelated to substrate selectivity, because we have shown through chimera constructions that the catalytic domain of Hrr25p is functionally similar to that of Yck2p. Rather, the specificity is conferred by the C-terminal region ofthese proteins. We suspect that the C-terminal region of CK1, which differs dramatically between isoforms, is responsible for the differential subcellular localization observed for different CK1 homologs. The subcellular distribution of the yeast CK1 homologs only partially parallels that described for individual isoforms of mammalian CK1. In human erythrocytes, CKIa is both cytosolic and plasma membrane-bound. Both forms are regulated by phosphatidylinositol 4,Fi-bisphosphate and phosphorylate cytoskeletal proteins in vitro (20, 21). In contrast, CKIa in Chinese hamster ovary cells and B82 mouse fibroblasts is localized to the spindle of mitotic cells, to the centromeres of interphase cells, and to endoplasmic reticulum (16). Thus, on CKIa does not appear to be the basis of subcellular ~stribution, the homolog of Ycklp, Yck2p, or Hrr25p. In addition, neither CKIa nor C a p is capable of Complernenting~c~~A~ck~A cells? On the basis of structure, theYck proteins are most similar to CKIy, whereas Hrr25p is most similar to Cm8. It is likely that additional mammalian isoforms await discovery. We conclude that members of the CKl family are localized to different subcellular compartments of the cell, where they fulfill different functions. This diversity of function and localization of CK1 enzymes is probably conserved in mammalian cells. Ackno~~edgmen~s-We thank Eric Chang, Ann Sutton, and Nancy Walworth for helpful advice, Mike Wigler and David G. Drubin for gifts of antibodies, Bob Derby and David Spector for assistance withmicroscopy, Spencer Teplin for oligonucleotide synthesis, and Jim DuQ, Michael Ockler, and Phil Rennafor art work and photography. B. Leichus, M. Cobb, and J. Kuret,unpublished

data.

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40.